Previous work from our laboratory indicates that NO and CO, two functionally-coupled molecules, confer robust and sustained protection against ischemia/reperfusion injury, via mechanisms that are unknown. NO donors are widely used clinically; CO donors have recently been developed by our collaborator, Dr. Motterlini, and may become a new class of anti-ischemic agents. The overall objective of Project 3 is to illuminate the molecular mechanisms that underlie the long-lasting infarct-sparing and anti-apoptotic effects observed after exposure of the heart to NO and CO. We propose that the NO-CO axis plays a critical role in the cardiac adaptation to stress by virtue of its ability to genetically reprogram the heart. Our fundamenta/hypothesis is that NO and CO act synergistically to induce a sustained cardioprotected phenotype by activating pro-survival signaling pathways via the recruitment of a cluster of stress-responsive transcription factors (STAT1, STAT3, and NF-kappaB), leading to the upregu/ation of anti-apoptotic proteins that inhibit both the death receptor- and the mitochondria-dependent pathways of apoptosis. In Aim 1 we will determine whether the sustained cardioprotective effects of NO and CO are due to inhibition of apoptosis vs. necrosis, which will be discerned with state-of-the-art techniques, including hairpin-1/caspase-3 and hairpin-2/caspase-3 dual-labeling assays. The influence of NO and CO donors on the extrinsic and intrinsic pathways of apoptosis will be elucidated by examining their effects on the release of cytochrome c and Smac, the activation of caspase-3, 8, and 9, and the expression of pro-apoptotic proteins (Bax, Bak, Bid, Bad) and anti-apoptotic proteins (Bcl-2, Bcl-XL, McI-1, c-FLIPL, c-FLIPs, ARC, XlAP, clAP-1, clAP-2). Aims 2 and 3 will establish whether the delayed anti-apoptotic and anti-necrotic actions of NO and CO are mediated by STAT1 and/or STAT3; this will be accomplished by knockout of the STAT1 gene and by genetic inhibition of STAT3 via constitutive cardiac-specific STAT3 knockout or via a novel inducible cardiac-specific STAT3 knockout. Aim 4 will determine whether the cardioprotective effects of the NO-CO axis require both Tyr-705 (via JAKs) and Ser-727 (via the PKCepsilon-Raf-1-MEK-1/2-p44/42 MAPK cascade) phosphorylation of STAT3, which will be interrogated by using a Tyr-705 dominant negative mutant of STAT3 and a Ser-727 mutant of STAT3, respectively. The role of PKCepsilon in the activation of the Raf-1-MEK1/2-p44/42 MAPK cascade will be interrogated with targeted deletion of PKCepsilon (PKCepsilon-/- mice). Using I-kappa-B-alpha dominant negative mutant (l-kappa-B-alpha-S32A,S36A mice), in Aim 5 we will determine whether (and the mechanism by which) NF-kappaB mediates the sustained cardioprotective effects of NO and CO. These studies will elucidate the specific roles of STAT1 vs. STAT3 and of NF-kappaB in myocardial ischemia/reperfusion injury. The results of this project will provide novel insights into the mechanism whereby the NO-CO axis genetically reprograms the heart, and will also have therapeutic implications for patients with coronary artery disease.